The development of a remote control device that can be used to manipulate neural activity in a brain sounds like the premise of a science fiction film, probably a summer blockbuster starring Keanu Reeves. However, the device is real, it stars a team of Berkeley Lab and UC Berkeley scientists, and it holds enormous promise for future studies into how the brain works.

Biophysicist Ehud Isacoff led the development of an artificial light-gated protein that provides a form of remote control over neural activity in the brain.

Ehud Isacoff, a biophysicist who holds joint appointments with Berkeley Lab's Physical Biosciences Division and UC Berkeley's Department of Molecular and Cell Biology, has led a study in which an artificial, light-activated protein was genetically engineered into the neurons of zebrafish. When a beam of light at a certain wavelength shines on the fish, it blocks their normal reflex response to a physical touch.

When the fish are illuminated with a second beam of light at a higher wavelength, the reflex response is restored. The engineered protein is called a "light-gated ionotropic glutamate receptor (LiGluR)" and was developed in a continuing collaboration between Isacoff and Dirk Trauner, a UC Berkeley chemistry professor.

"For the first time, we have the ability to target specific types of neurons within the neural circuits of a brain for selective activation," Isacoff says. "This ability to stimulate select neurons, either in isolated tissue or in living animals, is a big advantage for scientists seeking to determine how specific neuronal cell types contribute to brain functions and behavior."

The functions of the brain of any animal  human, fish, insect, whatever  are derived from the sending and receiving of electrochemical signals by various types of neurons that populate the brain and the central nervous system. When one neuron is sufficiently excited, it fires an electrical impulse, called an action potential (AP), which triggers the release of a neurotransmitter chemical across a synaptic junction to another neuron; this triggers the second neuron to fire an AP of its own. The synaptic connections of neurons consist of vast intercommunicating networks, which make possible motor, sensory, behavioral, and cognitive functions.

Until now, scientists investigating the links between neuron activity and brain functions have had to rely upon anatomical studies of neural wiring, either observing what happens when neural activities in local areas are disrupted or blocked, or observing the firing of neurons in various regions of the brain and associating this with specific functions.

"While these approaches have yielded important information about brain function, they all involve correlative analysis," Isacoff says. "It's widely acknowledged that such correlative approaches would be powerfully complemented by a method that can impose specific spatiotemporal patterns of activity on targeted neurons."

However, the manipulation of specific types of neurons in the intact nervous system has posed a difficult challenge. Every region of the brain contains complex circuits made up of many different kinds of neurons, and while electrical or magnetic techniques can be used to stimulate single neurons or local groups of neurons, such techniques are unable to target specific neuronal types. Likewise, flashes of light that activate small groups of neurons can rapidly release microsecond bursts of chemical transmitters, but these chemical transmitters are also generic to most kinds of neurons.

To provide the selectivity that is crucial to detailed studies of the brain, researchers have explored the idea of introducing into neurons a foreign receptor or channel that can be remotely controlled. A neuron is basically a high-speed transistor, which generates electrical impulses when ions cross the cell membrane through channels. Two years ago, Isacoff, Trauner, and their two research groups used a combination of genetic and chemical engineering techniques to alter an ion channel of central importance to neuroscience  the ionotropic glutamate receptor (iGluR). This channel plays a central role in allowing one neuron to pass its electrochemical signal to another.

Dirk Trauner is the UC Berkeley chemistry professor who oversaw the production of the LiGluR protein.

Isacoff and his colleagues added a new, light-activated chemical gate, synthesized in the Trauner lab, to the iGluR channel, creating what they dubbed LiGluR. When introduced into a neuron or other type of biological cell, the LiGluR functions as an optical switch, which can be used to turn on or turn off a specific biological activity within the cell.

"Optical switches are especially powerful elements in proteins, as they can be activated remotely with precise temporal and spatial control," Isacoff says. "Also, they are versatile, being applicable to any experimental situation in which you can deliver genes and light."

In their demonstration of the effectiveness of their LiGluR technology on neurons, Isacoff and his group first introduced the gene that encodes the engineered glutamate receptor into cultures of hippocampal neurons. They then exposed these cultures to the chemical light switch, which attaches covalently to the receptor protein. Flashes of violet light at a wavelength of 380 nanometers, lasting between one to five milliseconds  the same timescale as native neuronal activity  were found to depolarize the neurons so that their voltages became more positive, causing them to fire APs. This depolarization mimicked the synaptically evoked, excitatory postsynaptic potentials (EPSPs) that are normally generated by neurotransmitters released by another neuron. Illuminating the neurons with a second round of millisecond-length blue light, at a wavelength of 488 nanometers, immediately deactivated them.

"Our LiGluR also has the unique property that, once activated by a brief pulse of light, the channel will remain open in the dark until a pulse of deactivating light closes it," Isacoff says. "This makes it possible to evoke long depolarizations and trains of APs with minimal light exposure, another advantage for neuroscience research."

Demonstrating that the LiGluRs could also provide optical control over neuronal activity in vivo presented a significantly greater challenge. First there was the question of whether a transgenic organism could be engineered to express enough LiGluRs to influence neuronal activity. Then there were questions as to whether the expression of LiGluR would perturb behavior and whether it would be toxic to a living organism. Finally, there was the question of whether the chemical light switch would be able to penetrate the animal and reach the correct neurons.

Isacoff and his colleagues elected to work with the zebrafish, a tropical minnow that commonly serves as a model organism for genetic studies of vertebrates. The LiGluR gene was introduced and its expression could then be driven in different sets of neurons in the brain and spinal cord through a cross-breeding program. When the expression of the gene was in neurons that mediate touch sensation, the researchers tested their ability to control the touch response, a reflexive escape behavior that is triggered by mechanical pressure applied to the body, tail, or fins of the fish. Escape reflexes are critical to an organism's survival and are therefore among the strongest of all genetically hardwired behaviors.

Zebrafish, genetically modified to express the artificial protein LiGluR, were unable to execute their normal reflex response to a physical touch when illuminated by ultraviolet light. But the escape behavior was restored when the fish were illuminated with blue light.

"After illumination for about 15 minutes with light from a handheld UV lamp"  ultraviolet at a wavelength of 365 nanometers  "the behavior of virtually all of our transgenic larvae was altered," says Isacoff. "Optical activation of the LiGluR blocked the escape reflex so that the fish did not respond when touched with a pipette tip. The touch-response reflex was subsequently restored by illumination with blue light for 30 seconds."

To verify that the transgenes alone did not alter development or central nervous system functions of their zebrafish, Isacoff and his colleagues also tested the ability of the fish to free-swim, which requires an intact motor system, and to chase visual objects, a critical optomotor response. The results proved normal.

Isacoff says that LiGluR should provide similar control over other reflex responses in mice, rats and other animals. In principle, the technology should also be applicable to other types of inhibitory ionotropic receptors and other classes of neurons. Future plans for his research group will include the use of the LiGluR to study the formation of neural circuits and to investigate the neural bases of various behaviors.